Arichard:Notebook/bulk150210: Difference between revisions

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>Andrew
>Andrew
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==February 10, 2015 bulk nextera==
==February 10, 2015 bulk nextera==


Motivation: I want to test SM59 with our new IDT-Nextera v2 duel index primers against Nextera v1 with single index primers using bulk samples from 1-02 (AD) and 25-01 (CTL), but I don't have enough of all samples to compare everything at once. Therefore, my plan is to first extract DNA samples from the tubes I have in excess (cerebellum) to compare SM59 to Nextera v1, and wait on the more precious tubes (cortex) to use SM59.
Motivation: I want to make bulk libraries from 1-02 (AD) and 25-01 (CTL) using SM59 with our new IDT-Nextera v2 duel index primers, but I first need to test SM59 against 1:50 Nextera v1 with single index primers. Therefore, my plan is to first extract DNA samples from the tubes I have in excess (cerebellum) to compare SM59 to Nextera v1, and wait on the more precious tubes (cortex).


Goal: First, extract and freeze DNA from 1000 nuclei bulk samples from 1-02 and 25-01 cerebellum [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Samples/sort141009 sorted October 9, 2014]. Second, create libraries with SM59 dilutions and Nextera v1 and compare gels for intensity and distribution (tightest around ~300). Third, use best SM59 dilution to generate libraries from cortex.
Brandon already tested dilutions of SM59 and EZ-Tn5 (N = 1 per concentration) on HLS lysed cells (for accessibility), but it seems that the variability in lysis might be masking the dilution effect (no clear trend). He did include 6 ng purified genomic, which gave a very bright smear. I will use HLS-GHCl lysis followed by Ampure XP, pick one concentration of SM59 and Nextera v1 with more replicates.
 
Goal: First, to extract, purify, and freeze six 1000 nuclei bulk DNA samples from 1-02 cerebellum [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Samples/sort141009 sorted October 9, 2014]. Second, create libraries with 1X SM59 (Stock is 5X) and 1:50 Nextera v1 and compare gels for intensity and distribution. Third, use SM59 to generate bulk libraries from 1-02 and 25-01 cortex with N = 3 for all.


===Samples===
===Samples===
To test


{| {{table}}
{| {{table}}
Line 18: Line 22:
| align="center" style="background:#f0f0f0;"|'''ul HLS'''
| align="center" style="background:#f0f0f0;"|'''ul HLS'''
|-
|-
| 1-02 ctx full||1||120||3||1000||1||3||8.33||26.67
| 1-02 cbl full||4||274||10||1000||6||3||3.65||31.35
|-
|
|}
 
Remaining bulk libraries
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''sort'''
| align="center" style="background:#f0f0f0;"|'''# on tube'''
| align="center" style="background:#f0f0f0;"|'''nuclei/ul'''
| align="center" style="background:#f0f0f0;"|'''ul/tube'''
| align="center" style="background:#f0f0f0;"|'''nuclei/sample'''
| align="center" style="background:#f0f0f0;"|'''# samples'''
| align="center" style="background:#f0f0f0;"|'''# tubes to pool'''
| align="center" style="background:#f0f0f0;"|'''ul/sample'''
| align="center" style="background:#f0f0f0;"|'''ul HLS'''
|-
|-
| 1-02 ctx hi||2||120||3||1000||1||3||8.33||26.67
| 1-02 ctx full||1||120||3||1000||3||Not enough||NA||NA
|-
|-
| 1-02 ctx lo||3||78||10||1000||1||2||12.82||22.18
| 1-02 ctx hi||2||120||3||1000||3||Not enough||NA||NA
|-
|-
| 1-02 cbl full||4||274||10||1000||4||2||3.65||31.35
| 1-02 ctx lo||3||78||10||1000||3||4||12.82||22.18
|-
|-
| 25-01 ctx full||5||283||20||1000||4||1||3.53||31.47
| 25-01 ctx full||5||283||20||1000||3||1||3.53||31.47
|-
|-
| 25-01 cbl full||6||232||10||1000||4||2||4.31||30.69
| 25-01 cbl full||6||232||10||1000||3||2||4.31||30.69
|-
|-
|  
|  

Revision as of 23:41, 10 February 2015

February 10, 2015 bulk nextera

Motivation: I want to make bulk libraries from 1-02 (AD) and 25-01 (CTL) using SM59 with our new IDT-Nextera v2 duel index primers, but I first need to test SM59 against 1:50 Nextera v1 with single index primers. Therefore, my plan is to first extract DNA samples from the tubes I have in excess (cerebellum) to compare SM59 to Nextera v1, and wait on the more precious tubes (cortex).

Brandon already tested dilutions of SM59 and EZ-Tn5 (N = 1 per concentration) on HLS lysed cells (for accessibility), but it seems that the variability in lysis might be masking the dilution effect (no clear trend). He did include 6 ng purified genomic, which gave a very bright smear. I will use HLS-GHCl lysis followed by Ampure XP, pick one concentration of SM59 and Nextera v1 with more replicates.

Goal: First, to extract, purify, and freeze six 1000 nuclei bulk DNA samples from 1-02 cerebellum sorted October 9, 2014. Second, create libraries with 1X SM59 (Stock is 5X) and 1:50 Nextera v1 and compare gels for intensity and distribution. Third, use SM59 to generate bulk libraries from 1-02 and 25-01 cortex with N = 3 for all.

Samples

To test

sort # on tube nuclei/ul ul/tube nuclei/sample # samples # tubes to pool ul/sample ul HLS
1-02 cbl full 4 274 10 1000 6 3 3.65 31.35

Remaining bulk libraries

sort # on tube nuclei/ul ul/tube nuclei/sample # samples # tubes to pool ul/sample ul HLS
1-02 ctx full 1 120 3 1000 3 Not enough NA NA
1-02 ctx hi 2 120 3 1000 3 Not enough NA NA
1-02 ctx lo 3 78 10 1000 3 4 12.82 22.18
25-01 ctx full 5 283 20 1000 3 1 3.53 31.47
25-01 cbl full 6 232 10 1000 3 2 4.31 30.69

Protocol

Following protocol from January 22 up to Nextera

Lysis and freezedown

  1. Lyse in 35 ul 1X HLS at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
  2. 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
  3. Elute in 7 ul H2O